Abstract
The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink both in vitro and in vivo. Five Pseudomonas aeruginosa (P. aeruginosa) strains were isolated from lungs of mink with suspected hemorrhagic pneumonia and their identity was confirmed by morphological observation and 16S rDNA sequence analysis. Compared to P. aeruginosa strains isolated from mink with hemorrhagic pneumonia in 2002, these isolates were more resistant to antibiotics selected. A lytic phage vB_PaeP_PPA-ABTNL (PPA-ABTNL) of the Podoviridae family was isolated from hospital sewage using a P. aeruginosa isolate as host, showing broad host range against P. aeruginosa. A one-step growth curve analysis of PPA-ABTNL revealed eclipse and latent periods of 20 and 35 min, respectively, with a burst size of about 110 PFU per infected cell. Phage PPA-ABTNL significantly reduced the growth of P. aeruginosa isolates in vitro. The genome of PPA-ABTNL was 43,227 bp (62.4% G+C) containing 54 open reading frames and lacked regions encoding known virulence factors, integration-related proteins and antibiotic resistance determinants. Genome architecture analysis showed that PPA-ABTNL belonged to the “phiKMV-like Viruses” group. A repeated dose inhalational toxicity study using PPA-ABTNL crude preparation was conducted in mice and no significantly abnormal histological changes, morbidity or mortality were observed. There was no indication of any potential risk associated with using PPA-ABTNL as a therapeutic agent. The results of a curative treatment experiment demonstrated that atomization by ultrasonic treatment could efficiently deliver phage to the lungs of mink and a dose of 10 multiplicity of infection was optimal for treating mink hemorrhagic pneumonia. Our work demonstrated the potential for phage to fight P. aeruginosa involved in mink lung infections when administered by means of ultrasonic nebulization.
Highlights
The mink industry has been growing in production with more than 50 million mink pelts harvested globally in 2010 (European Fur Breeder’s Association (EFBA) Annual Report 2010)
In the LB agar medium, the purified bacterial isolations formed colonies which showed characteristics of round shape, convex surface, smooth margin, viscous and moist texture. They all produced a blue-green pigment in LB liquid medium. These isolates were named after their sample numbers, such as PA1-1, PA5-1-1, PA5-1-2, PA5-2-1 and PA5-2-2. 16S rDNA fragments from all isolates were amplified by PCR using universal primers
BLAST analysis showed that the partial 16S rDNAs of the above five strains were more than 98% identical with that of P. aeruginosa strains RHH13, RHH13, JL 091016, D1, JL091016 (GenBank accession number: HQ143612.1, HQ143612.1, HM224410.1, KF113578.1 and HM224410.1), respectively
Summary
The mink industry has been growing in production with more than 50 million mink pelts harvested globally in 2010 (European Fur Breeder’s Association (EFBA) Annual Report 2010). The high animal density commonly associated with mink farming presents a great opportunity for the spread of diseases [1]. Hemorrhagic pneumonia, caused by Pseudomonas aeruginosa (P. aeruginosa), has been one of the most costly infectious diseases among farmed mink [2,3]. Mink of all ages are affected with mortality approaching 50% in some affected farms [5]. Antibiotics such as gentamycin, polymyxin and penicillinare mainly used to treat hemorrhagic pneumonia in mink [6]. Multivalent vaccines against mink hemorrhagic pneumonia were available, application of these commercial vaccines was limited because of their exorbitant price and short protection period [5]. Other therapeutic approaches are being explored, as new antibacterial compounds are scarce [8]
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